The replication defective Spleen Focus-Forming Viruses (SFFV) have been implicated in the pathogenesis of acute erythroleukemia induced by stocks of the Friend and Rauscher murine leukemia viruses (MuLV). The available evidence suggests that the expression of an envelope glycoprotein designated gp52 is a prerequisite for disease production by SFFV. The gp52 glycoprotein is defective in its transport to the cell surface, and the resulting intracellular accumulation of this molecule may play a role in pathogenicity. We wish to determine the molecular features that account for the defective intracellular transport of SFFV gp52 and establish whether the intracellular accumulation of gp52 is a prerequisite for the expression of Friend disease. An analysis of the nucleotide sequence of the env genes of SFFV and related replication competent MuLV indicates that SFFV gp52 is related to the envelope proteins of recombinant mink cell focus-forming (MCF) virus with the following additional changes: i) a 585-bp large deletion in the gp70-p15E junctional region of the molecule; ii) insertion of a 6-bp tandem repeat in the membrane spanning region of p15E; and iii) a single base insertion also in the membrane spanning region of p15E which alters the reading frame to generate a termination codon, 34 codons prior to the original stop codon. We propose to examine the effects of specific changes in the SFFV genome using oligonucleotide directed site-specific mutagenesis and exchange of restriction fragments from Friend MuLV or Friend MCF viruses such that one or more of the changes in SFFV as compared to the MuLV are corrected. NIH 3T3 cells will be transformed with these reconstructed SFFV genomes with or without helper MuLV genomes to establish SFFV-infected cells. The subcellular distribution and intracellular transport of the reconstructed gp52 molecules will be studied by biochemical and electron microscopic techniques. These results should establish which of the different changes involved in the generation of gp52 is responsible for its defective intracellular transport. The mutant SFFV genomes will be rescued from the transformed cells as pseudotypes with Moloney MuLV, and the pathogenicity of these M-MuLV/SFFV pseudotypes will be studied by splenic focus assay in mice.